During this reporting period, the Laboratory of Genetics and Physiology has made progress and elucidated molecular mechanisms that enable mammary super-enhancers to sense cytokines during pregnancy and that ensure their accurate spatial control. Notably, we have identified, for the first time, distinct mechanisms used by super-enhancers and regular enhancers to activate their respective target genes in response to cytokines. We have also identified that putative chromatin boundaries established by the protein CTCF have a limited capacity to shield super-enhancers from non-target genes. Lastly, we have conducted a large genetic study and determined that CRISPR-Cas9 genome editing is an efficient tool to introduce deletions in regulatory elements in the mouse genome. However, we observed an unexpected extent of unwarranted deletions and insertions of DNA that needs to be taken into account when considering this technology. In a quest to understand how cytokine-sensing enhancers and super-enhancers control common and lineage-specific genes we focused on the regulation of the entire genome and two genes in particular, Socs2 and Wap (ref. 7). While Wap is expressed exclusively in mammary epithelium and is induced by prolactin, Socs2 is expressed in many, if not all, cell types and activated by a plethora of cytokines, including several interleukins, prolactin and growth hormone. These cytokines activate the transcription factor STAT5 that is a principal regulator of enhancers and super-enhancers driving these two genes. Here have demonstrated that STAT5 activates lineage-specific and widely expressed genes through different mechanisms. STAT5 preferentially binds to promoter sequences of cytokine-responsive genes expressed across cell types and to putative enhancers of lineage-specific genes. While chromatin accessibility of STAT5-based enhancers was dependent on cytokine exposure, STAT5-responsive promoters of widely expressed target genes were generally constitutively accessible. Although the contribution of STAT5 to enhancers is well established, its role on promoters is poorly understood. To addressed this question using the Socs2 gene as a paradigm. Upon deletion of STAT5 response elements from the Socs2 promoter in the mouse genome, cytokine induction was abrogated, while basal activity remained intact. Our studies suggest that promoter-bound STAT5 modulates cytokine responses and enhancer-bound STAT5 is mandatory for gene activation. Cytokines utilize the transcription factor STAT5 to control cell-specific genes at a larger magnitude than universal genes, with a mechanistic explanation yet to be supplied. We addressed this conundrum by investigating enhancers and super-enhancers in the mammary gland, tissue rich in STAT5 due to an autoregulatory loop of the Stat5 gene PMID: 26446995). Using genome-wide studies we have identified putative STAT5-based mammary-specific and universal enhancers, which served as an entry point to investigate mechanisms underlying their differential response to cytokines. We have interrogated the integrity and function of both categories of regulatory elements using biological and genetic approaches (ref. 6). During lactation, STAT5 occupies mammary-specific and universal cytokine-responsive elements. Following lactation, prolactin levels decline and while mammary-specific STAT5-dependent enhancers are decommissioned with 24 hours, universal regulatory complexes remain intact. These differential sensitivities are linked to STAT5 concentrations and the mammary-specific Stat5 autoregulatory enhancer. In its absence, mammary-specific enhancers, but not universal elements, failed to be fully established. Upon termination of lactation STAT5 binding to a subset of mammary enhancers was substituted by STAT3. No STAT3 binding was observed at the most sensitive STAT5 enhancers suggesting that upon hormone withdrawal their chromatin becomes inaccessible. Lastly, we have demonstrated that the mammary-enriched transcription factors GR, ELF5 and NFIB associate with STAT5 at sites lacking bona fide binding motifs. This study (ref. 6) provided, for the first time, molecular insight into the differential sensitivities of mammary-specific and universal cytokine-sensing enhancers. Cytokine-sensing super-enhancers are among the most powerful regulatory elements controlling their respective target genes without affecting other nearby genes. There is a gap in knowledge in the mechanisms used to ensure that super-enhancers do not inadvertently activate non-target genes. The zinc finger protein CTCF has been invoked in establishing boundaries between genes, thereby controlling spatial and temporal enhancer activities. However, there is limited genetic evidence to support the concept that these boundaries restrict the search space of enhancers. We have addressed this question in the casein locus containing five mammary and two non-mammary genes under the control of at least seven putative enhancers (ref. 3). We have identified two CTCF binding sites flanking the locus and two associated with a super-enhancer. Individual deletion of these sites from the mouse genome did not alter expression of any of the genes. However, deletion of the border CTCF site separating the Csn1s1 mammary enhancer from neighboring genes resulted in the activation of Sult1d1 at a distance of more than 95 kb but not the more proximal and silent Sult1e1 gene. Loss of this CTCF site led to de novo interactions between the Sult1d1 promoter and several enhancers in the casein locus. Our study demonstrated that only one out of the four CTCF sites in the casein locus had a measurable in vivo activity. Precise spatiotemporal gene regulation is paramount for the establishment and maintenance of cell-specific programs. Although there is evidence that chromatin neighbourhoods, formed by the zinc-finger protein CTCF, can sequester enhancers and their target genes, there is limited in vivo evidence for CTCF demarcating super-enhancers and preventing cross talk between distinct regulatory elements. To further investigate the biological roles of CTCF and their binding sites in restricting super-enhancer activities to their respective target genes, we studied the Wap locus that is under the control of a super-enhancer, which activates the Wap gene more than 1,000-fold during pregnancy (ref. 5). The Wap locus with its mammary-specific super-enhancer is separated by CTCF sites from widely expressed genes. Mutational analysis demonstrated that the Wap super-enhancer controls Ramp3, despite three separating CTCF sites. Their deletion in mice resulted in elevated expression of Ramp3 in mammary tissue through elevated promoter-enhancer interactions. Deletion of the distal CTCF binding site resulted in loss of Ramp3 expression in non-mammary tissues. This suggests that CTCF sites are porous borders, allowing a super-enhancer to activate a secondary target. Likewise, CTCF sites shield a widely-expressed gene from suppressive influences of a silent locus. Over the past few years CRISPR/Cas9 genome editing has become the premier tool to interrogate the genome through the introduction of mutations (deletions, insertions and point mutations). Although this technology has provided unprecedented opportunities to interrogate the functional significance of any given genomic site, there is a paucity of data on the extent of molecular scars inflicted on the mouse genome. Understanding the degree to which CRISPR/Cas9 genome editing affects target sites in the genome is highly relevant for the interpretation of studies based on this technology. To address this important issue we have interrogated the molecular consequences of CRISPR/Cas9-mediated deletions at 17 sites in four loci of the mouse genome (ref. 4). We have sequenced targeted sites in 632 founder mice and analyzed 54 established l